PROJECT SUMMARY Resident microbes of the gastrointestinal (GI) tract have wide and powerful effects on many aspects of host physiology. These resident microbes, the gut microbiota, act through the intestinal lumen to influence host physiology by both direct and indirect mechanisms. Emerging evidence suggests that alterations in the interaction between host and microbes influence many diseases prevalent in modern society, including obesity and its metabolic sequelae. Despite the prevalence of obesity, broadly effective therapies for this disease have been elusive. Roux-en-Y gastric bypass (RYGB) and related bariatric surgical procedures are currently the most effective therapies for obesity and related metabolic disorders. Metabolic improvements resulting from these operations can be attributed, at least in part, to alterations in the gut microbiome. Transfer of microbiota derived from both mice and human patients having undergone RYGB induces a significant portion of the metabolic phenotypes of surgery. However, the identity of the microbes, and/or their metabolites that regulate body weight change, is/are unknown. We propose to examine host-microbe interactions within the context of bariatric surgery to begin to establish a functional link between microbes and metabolic changes in the host. The overall goal of the proposed studies is to create and validate a system to study critical interactions between the microbiota, their products, and the host gastrointestinal mucosa, which can then be used to screen and identify physiologically important and luminally active molecules that regulate metabolism. To do so, we will identify a component of the RYGB-specific gene expression signature that (a) is recapitulated in vivo by RYGB-induced changes in the microbiota, (b) persists in enteroid culture models, and (c) can be induced in vitro by microbiota, cultured bacteria or their metabolic products. In Aim 1, we will use fecal transplantation from a mouse model of RYGB to a germ-free diet-induced obese (DIO) mouse model to identify a transcriptional signature in the GI mucosa that is induced by RYGB microbiota. In Aim 2, we will examine the overlap in gene expression changes between the in vivo mouse model of RYGB and in vitro intestinal epithelial organoids (enteroids) derived from surgical animals to validate how the enteroid model recapitulates the in vivo transcriptional profile. In Aim 3, we will add microbes and their products from RYGB mice to DIO-derived enteroids to identify a subset of RYGB-induced changes in vitro. These changes will provide the basis for an in vitro assay system to identify microbes that modulate metabolism. From these studies, we will gain an understanding of how microbes influence host transcription in vivo and in vitro and how well enteroids recapitulate in vivo biology. The results of this study will provide the basis for future work on how microbes affect host transcription to exert metabolic changes.